Dec 20, 2025
hPBMCs thawing and plating (no specific down-stream assay)
- Rebecca Wallings1,
- Malu Tansey1
- 1Indiana University
Protocol Citation: Rebecca Wallings, Malu Tansey 2025. hPBMCs thawing and plating (no specific down-stream assay). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj1ow5vk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 20, 2025
Last Modified: December 22, 2025
Protocol Integer ID: 235487
Keywords: ASAPCRN, human pbmcs from cyrovial, plating human pbmc, hpbmc, stream assay, cyrovial
Funders Acknowledgements:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020527
Abstract
General protocol for thawing and plating human PBMCs from cyrovials.
Materials
- Water bath at 37 degrees (use on in main lab)
- Dry ice
- 1 x 50mL falcon tube per cryovial, with 25mL of thawing media at 37 degrees
- MACs buffer at 37 degrees
- Thawing media at 37 degrees
- Plating media at 37 degrees
- Tissue culture plasticware: 24-well plates, p1000 pipette tips, p20 pipette tips, stripettes, aspirating stripettes, trypan blue, countess slides, eppendorfs
Troubleshooting
Protocol
Retrieve cryovials for plating from Mr W and place on dry ice.
Make sure you have 1 falcon with thawing media per cryovial ready in the hood, pre-warmed to 37 degrees.
Place cells in floaty and place in water bath and allow to thaw for approx. 90 seconds.
Check to see if thawed, a small amount of ice remaining is fine.
Pour contents of cryovial into falcon containing thawing media.
Centrifuge falcons at room temp for 10 minutes at 300 x g.
Aspirate supernatant and resuspend pellet in 10mL MACs buffer – make sure resuspend pellet first in 1mL of warmed MACs with a p1000 pipette to ensure pellet is broken up, then add 9mL MACs with stripette.
Take 10uL of cell suspension and place in Eppendorf.
Centrifuge falcons 300 x g 10 mins at room temp.
Whilst falcons are centrifuging proceed with cell count: mix 10uL of trypan blue with 10uL of cell suspension, place 10uL in both chambers of a countess slide and count using “hPBMC cryo” programme on countess.
Note cell count per mL (countess accounts for 1:1 trypan blue dilution, so this number is accurate already) and live cell % (aiming for 80+ % viability).
Calculate volume of plating media needed for each pellet to obtain 1 x 10^6^ cells per mL.
Example: countess count = 5 x 10^5^ per mL. 5 x 10^5^ x 10 (as cells were in 10mL) = 5 x 10^6^ cells total. Therefore resuspend cells in 5mL for 1 x 10^6^ per mL.
For each patient sample, plate 500uL of cell suspension per well of a 24-well plate, and plate 1 well per treatment required for experiment.
NOTE: This can be adjusted based on downstream assay being used. E.g. flow cytometry = 1 million/mL and plate 500uL in 24 well plate (results in 500,000 per well), whilst RNA needs more cells so plate 2mL in 6 well plate (results in 2 million per well).
For each patient, label wells with patient ID and treatments.
Place in incubator for MINIMUM of 2 hours to rest and proceed with downstream assay.